Gene name - charybde and scylla
Synonym - charybdis (char)
Cytological map positions - 68C
Function - unknown
Keywords - negative regulation of growth, insulin pathway, response to hypoxia
Symbol - chrb and scyl
Genetic map position - 3L
Classification - conserved protein
Cellular location - cytoplasmic and nuclear
Diverse extrinsic and intrinsic cues must be integrated within a developing organism to ensure appropriate growth at the cellular and organismal level. In Drosopohila, the insulin receptor/TOR/S6K signaling network plays a fundamental role in the control of metabolism and cell growth. scylla and charybdis (a. k. a. charybde), two homologous genes identified as growth suppressors in an EP (enhancer/promoter) overexpression screen, act as negative regulators of growth. The genes are named after mythological monsters said to have lived in the Strait of Messina between Sicily and Italy, that posed a threat to the passage of ships. The simultaneous loss of both genes generates flies that are more susceptible to reduced oxygen concentrations (hypoxia) and that show mild overgrowth phenotypes. Conversely, either scylla or charybdis overactivation reduces growth. Growth inhibition is associated with a reduction in S6K but not PKB/Akt activity. Together, genetic and biochemical analysis places Scylla/Charybdis downstream of PKB and upstream of TSC1. Furthermore, scylla and charybdis are induced under hypoxic conditions and scylla is a target of Drosopohila HIF-1 (hypoxia-inducible factor-1: Similar) as is its mammalian counterpart RTP801/REDD1, thus establishing a potential cross-talk between growth and oxygen sensing (Reiling, 2004).
The evolutionarily conserved Insulin/IGF receptor (Inr)/Target of Rapamycin (TOR) signaling network plays an important role in modulating growth, metabolism, reproduction, and life span in response to intracellular and extracellular signals in species ranging from invertebrates to humans. In Drosopohila, viable mutant combinations of positive components of the Drosopohila Inr cascade such as Inr, chico (the homolog of vertebrate IRS1-4), PKB (Protein kinase B, also known as Akt) and PDK1 (3-phosphoinositide-dependent protein kinase-1) lead to developmentally delayed and proportionally reduced small flies, displaying a reduction in cell size and number. In contrast, loss of PTEN (phosphatase and tensin homolog on chromosome ten), which antagonizes PI3K activity by dephosphorylating the 3'-position of phosphoinositides, leads to hypertrophy and hyperplasia. In humans, loss of the tumor suppressor PTEN is observed frequently in glioblastomas, prostate cancers, and endometrial cancers, and PTEN germline mutations are linked to dominant hamartoma syndromes like Cowden syndrome, Lhermitte-Duclose disease, Proteus syndrome, and Bannayan-Zonana syndrome. Genetic studies in Drosophila indicate that PKB has a crucial role in signaling downstream of PTEN since flies completely lacking PTEN function can be rescued to viability by lowering PKB activity (Reiling, 2004 and references therein).
The TOR/S6K (S6 kinase) branch of the growth modulatory network is negatively regulated by the tumor suppressor Tsc1 (hamartin)/Tsc2 (tuberin: Drosophila homolog is Gigas) complex. Tuberous sclerosis complex (TSC) is an autosomal-dominant disorder characterized by the formation of hamartomas, benign tumors that arise in various tissues. In Drosophila, cells devoid of Tsc1/Tsc2 function are increased in size. Tsc2 and, more weakly, Tsc1 were found to physically associate with dTOR, thereby inhibiting dTOR kinase activity. Other studies reported an inhibitory role of PKB on Tsc1-Tsc2 by PKB-mediated phosphorylation of Tsc2. Recently, the small GTPase Rheb (Ras homolog enriched in brain) has been identified as a new positive growth effector acting downstream of Tsc1-Tsc2 and upstream of TOR. Mechanistically, Tsc2 acts as GTPase-activating protein (GAP) toward Rheb. The molecular mechanism to explain how Rheb relays the signal to TOR is currently unknown. dTOR mutants show a growth deficit that is more pronounced in endoreplicative tissues than in mitotic tissues. An effector of mTOR is S6K, which upon activation by mTOR phosphorylates ribosomal protein S6. S6K-mediated S6 phosphorylation has been thought to lead to a preferential translation of mRNAs encoding ribosomal proteins and proteins of the translational apparatus although the significance of this S6K function has been questioned. Inr/TOR signaling activity culminates in the regulation of translation rate by controlling S6K and the translational repressor 4E-BP1. S6K mutant flies are small but in contrast to mutants of the Inr pathway, only cell size is reduced without a change in cell number. Therefore, loss of S6K function reduces growth and body size to a lesser extent than loss of other positive components acting further upstream in the cascade (Reiling, 2004).
Growth is modulated by extrinsic factors such as nutrients, temperature, and hypoxia. However, their link to the Inr/TOR signaling network is not well defined. Although it is known that starvation results in a reduction in the levels of insulin-like peptides and a reduction in S6K activity in Drosopohila, little is known about whether temperature or hypoxia regulates the activity of this pathway. It is conceivable that mutations in genes coding for factors mediating the modulation of growth in response to external stimuli have escaped detection because they do not exhibit a very strong phenotype under standard culture conditions. For example, overexpression of the Forkhead transcription factor FOXO3a, the human homolog of Drosopohila dFOXO, produces a very subtle small eye phenotype under normal nutrient conditions. Under starvation, however, this phenotype is massively exacerbated, inducing a further eye size reduction and cell death. Furthermore, dFOXO mutant flies are viable and do not show an (over)growth phenotype in an otherwise wild-type background under normal conditions. Genes like dFOXO were missed in genetic loss-of-function screens aimed at identifying growth-regulatory genes (1) because they have only mild or missing mutant phenotypes under normal conditions and/or (2) because their function is masked by redundancy (Reiling, 2004).
Genes acting in a redundant manner can be identified in a complementary gain-of-function approach using EP (enhancer/promoter) elements. Screening >4000 novel EP lines, two insertions were found in the scylla locus as suppressors of a PKB/PDK1-dependent eye overgrowth phenotype. A second gene was identified in the Drosopohila genome with homology to scylla, and named charybdis. Homologs of these genes also exist in mammals, and they have been implicated in the response of a cell to hypoxia or more generally as stress-induced genes having either pro- or anti-apoptotic functions. Evidence is presented that scylla and charybdis, like some of their mammalian homologs, are induced by hypoxia and that Scylla and Charybdis act as partially redundant negative regulators of growth by controlling S6K but not PKB activity (Reiling, 2004).
Therefore the two related proteins, Scylla and Charybdis, are negative modulators of Inr/TOR signaling in response to different external stress situations including hypoxia and starvation. scylla and charybdis single mutants do not show obvious growth phenotypes. scylla charybdis double-mutant flies are also viable and fertile and exhibit a slight increase in body size. Viability of the double mutants is strongly reduced, however, when they are reared under hypoxic conditions. Thus, although Scylla and Charybdis are largely dispensable for normal development, they have a critical role for the endurance of prolonged hypoxia. scylla is transcriptionally induced as a target of Drosopohila HIF-1 (the Tango-Similar dimer) and that scylla and charybdis are up-regulated under hypoxic conditions like their mammalian homolog RTP801/REDD1. Furthermore, Scylla negatively regulates Inr/TOR signaling by reducing S6K but not PKB activity (Reiling, 2004).
RTP801 was described as a hypoxia/HIF-1-inducible factor with a role in apoptosis (Shoshani, 2002). scylla/charybdis, like their mammalian homolog, are induced by hypoxia, and scylla is a direct target of Drosopohila HIF-1. However, there is no indication that scylla/charybdis overexpression promotes cell death. Coexpression of the caspase inhibitors p35 or DIAP1 did not rescue the small eye phenotype induced by expression of either scylla or charybdis in the eye. Moreover, acridine orange staining revealed no increased cell death upon scylla/charybdis coexpression. Accordingly, signs of necrotic eye tissue were never observed upon scylla/charybdis overexpression (Reiling, 2004).
Dig2, a murine Scylla/Charybdis homolog, is also a stress-responsive protein induced by a variety of treatments including dexamethasone, thapsigargin, tunicamycin, and heat shock (Wang, 2003). Together with the finding by Zinke (2002) that scylla/charybdis expression is increased during starvation conditions and the current analysis, showing that Scylla and Charybdis act as growth inhibitors, these data support a model wherein Scylla and Charybdis, induced by stresses like hypoxia and starvation, act to dampen growth under certain stress conditions (Reiling, 2004).
Overexpressing either scylla or charybdis on their own is sufficient to reduce growth. Coexpression of both proteins seemed to have a slight cooperative effect on the PKB/PDK1-dependent eye phenotype with respect to ommatidial structure. Thus, an obvious question is whether Scylla and Charybdis bind to each other and exert their effect only in the presence of the other. Notably, eye-specific charybdis overexpression in a scylla-/- background results in the same phenotype as in a wild-type situation. This indicates that Charybdis can act independently of Scylla. This is further supported by their mostly nonoverlapping mRNA expression patterns. Indeed, charybdis but not scylla is expressed in neuromuscular junctions. Moreover, under hypoxia only scylla is induced in the fat body but not charybdis, indicating that they may be differentially regulated (Reiling, 2004).
Several lines of evidence indicate that Scylla and Charybdis feed into the Inr pathway downstream of PKB: (1) Scylla antagonizes PKB/PDK1-induced overgrowth in the eye, but in vitro kinase assays demonstrate that Scylla and Charybdis do not reduce PKB kinase activity, nor does the loss of Scylla enhance PKB kinase activity. (2) scylla or charybdis coexpression can rescue PKB-induced developmental lethality, and ubiquitous scylla expression suppresses the lethality associated with hypomorphic PTEN mutants. (3) The weight reduction of hypomorphic PKB3 flies is partially rescued by the simultaneous absence of Scylla function. (4) Eye-specific PKB/PDK1 expression in a scylla-/- background leads to a mild enhancement of the eye phenotype. (5) Overexpression of PTEN or Dp110DN does not suppress the big eye phenotype of the tester system, suggesting that the screening system identifies components that act downstream of PKB (Reiling, 2004).
S6K assay has demonstrated that Scylla is capable of reducing S6K activity. Thus, Scylla acts upstream of S6K. This result is consistent with in vivo results. (1) scylla overexpression in the wing reduces wing size by decreasing cell size but not cell number (in fact, cell number is slightly increased), and (2) a S6K scylla mutant combination has the same weight as S6K single mutants. S6K mutants are smaller because of a reduction in cell size but not cell number, making it distinct from other Inr signaling pathway mutants. Scylla and Charybdis do not control S6K activity directly but require its upstream regulator TSC. Tsc1/2 mutants cannot be rescued by overexpression of scylla, and the big head phenotype caused by loss of TSC function is not enhanced by the absence of Scylla and Charybdis. Coexpression of scylla or charybdis and Tsc1/2 does not further decrease eye size compared to Tsc1 and Tsc2 co-overexpression on their own. Moreover, a Rheb-dependent big eye phenotype or lethality induced by ubiquitous Rheb expression cannot be suppressed by scylla expression. These results indicate that Scylla regulates S6K activity by acting upstream of Tsc1/2 and Rheb. This function appears to be conserved between mammals and flies because RTP801/REDD1 (Brugarolas, 2004) can reduce S6 phosphorylation only in the presence of TSC (Reiling, 2004).
The findings indicate that scylla/charybdis overexpression mainly affects the TSC/TOR/S6K branch of the pathway downstream of PKB. The PKB-FOXO axis appears not to be influenced by Scylla and Charybdis. Eye-specific overexpression of scylla/charybdis in conjunction with FOXO is unable to induce 4EBP. In contrast, simultaneous expression of FOXO and PTEN or a dominant-negative form of PI3K leads to a strong induction of the reporter gene (Reiling, 2004).
Consistent with an interplay between the Inr and TOR/S6K pathways, Inr lethality is suppressed by heterozygosity of Tsc1. Furthermore, overexpressed PKB phosphorylates and inactivates Tsc2 and thereby activates S6K. The finding that scylla overexpression is sufficient to rescue the lethality associated with PKB overexpression indicates that the lethality caused by PKB overexpression is due to the hyperactivation of the TOR/S6K pathway. Thus, oncogenic activation of PI3K/PKB signaling seems to be mainly mediated by TOR/S6K signaling. This may explain the beneficial effect of Rapamycin treatment (or its derivatives CCl-779 and RAD001) on PTEN-deficient tumors or cells overexpressing PKB (Reiling, 2004 and references therein).
TSC and TOR receive multiple inputs reflecting the metabolic state of the cell. AMP-activated kinase (AMPK) is a heterotrimeric kinase that is activated by high AMP/ATP ratios in the cell. ATP depletion induces Tsc2-phosphorylation, and it was found that AMPK could interact with and phosphorylate Tsc2. Interestingly, loss of Tsc2 in MEFs and U2OS osteosarcoma cells under low serum and prolonged hypoxia conditions results in HIF-1alpha accumulation and concomitantly increased expression of HIF-1 targets in a Rapamycin-dependent manner. It has been shown that mTOR is regulated by decreased oxygen concentration resulting in a dephosphorylation of mTOR at Ser 2481, an mTOR autophosphorylation site. This effect is accompanied by reduced S6K phosphorylation but does not correlate with changes in adenine nucleotide levels and AMPK phosphorylation. Hence, these findings suggest a role for AMPK/Tsc2/mTOR in the integration of oxygen sensing/energy metabolism and growth (Reiling, 2004).
Frei (2004) found mutations in the gene encoding Drosopohila HIF-1 prolyl hydroxylase (Hph), the enzyme rendering HIF-1alpha a substrate for proteasomal destruction under normoxic conditions, function as dominant suppressors of a Cyclin D/Cdk4-induced bulging eye phenotype. Cells defective for hph show a growth deficit, and its overexpression stimulated growth. This study suggested that the growth-promoting function of Hph is independent of HIF-1alpha/Sima. The results raise the possibility that the Sima target scylla is important under hypoxia for growth inhibition (Reiling, 2004).
Directed expression of Tgo-Sima in the fat body induces scylla expression. That this regulation is physiologically relevant can be inferred from three findings: (1) scylla is also induced under hypoxic conditions; (2) directed expression of other bHLH-PAS proteins like Tgo-Trh or Sim alone did not induce scylla expression; (3) survival of flies lacking scylla and charybdis function is severely compromised under hypoxic conditions. It is suggested that scylla and charybdis are induced in response to external stress stimuli (e.g., hypoxia and starvation) to inhibit growth downstream of PKB but upstream of Tsc1/2. Scylla suppresses growth by reducing S6K activity. This could be achieved by relieving the inhibitory effect of PKB on Tsc2. Alternatively, Scylla/Charybdis could be negatively regulated targets of PKB. This is unlikely, however, since Scylla and Charybdis lack PKB consensus phosphorylation sites. AMPK, activated by drops in energy levels, may also contribute to the induction process of scylla and charybdis for growth inhibition, presumably under prolonged stress exposure. However, it is also possible that AMPK is controlled by Scylla and/or Charybdis. AMPK decreases protein synthesis by inhibition of S6K in a Rapamycin-sensitive manner, suggesting that mTOR is involved in mediating AMPK signaling (Kimura, 2003). AMPK also phosphorylates Tsc2, an event important for the cellular energy response pathway (Reiling, 2004 and references therein).
In tumors, hypoxic microenvironments are often encountered. Tumor hypoxia is associated with poor prognosis and resistance to radiation-induced cell death. Mutations in the tumor suppressor von Hippel-Lindau (VHL), the subunit of an E3 ubiquitin ligase complex that recognizes proline-hydroyxlated residues in HIF-1alpha, led to the formation of a variety of tumors including clear cell carcinomas of the kidney, pheochromocytomas, and hemangioblastomas. VHL-defective tumors exhibit increased HIF-1alpha expression. The induction of RTP801/REDD1 in cells exposed to hypoxia in tumors raises the possibility that these genes may play a role in tumor development. RTP801/REDD1 may act as a tumor suppressor. Cells having lost RTP801/REDD1 function may not stop growing under hypoxic conditions and hence risk accumulating further mutations that promote their tumorigenic state. The analysis of RTP801/REDD1 expression or mutations in a variety of tumor cell lines should help to test this hypothesis (Reiling, 2004).
scylla codes for a 280-amino acid polypeptide with a predicted molecular weight (MW) of ~31 kDa, and the Charybdis protein contains 299 amino acid residues (MW ~32 kDa). Since EP9.85 is located in the coding region of scylla, thus generating a Scylla protein with an N-terminal truncation of 12 amino acids, tests were performed to see whether a UAS-scy transgene encoding the truncated form of Scylla behaves as the full-length protein. Overexpression of UAS-scyDelta1-12 and UAS-scywt showed the same effects as EPscy, indicating that amino acids 1-12 are dispensable for the growth-suppressing function of Scylla (Reiling, 2004).
Homologs of Scylla/Charybdis exist throughout the animal kingdom including humans, rat, mouse, Xenopus, and zebrafish. Reminiscent of the situation in Drosopohila, mammals possess several paralogous proteins with homology to Scylla/Charybdis. At least two homologs (RTP801/REDD1 and FLJ3691, a hypothetical protein presumably corresponding to REDD2 (Ellisen, 2002) exist in humans, two (RTP801 and SMHS1) in rat, and three (RTP801L, Dig2, and SMHS1) in mice. The overall homology is highest toward the C terminus. Charybdis contains a predicted coiled-coil structure (amino acids 168-188) (Reiling, 2004).
In mammals, RTP801/REDD1 and other members of the family are induced upon various forms of cellular stress including hypoxia, DNA damage, and dexamethasone treatment (Ellisen, 2002; Shoshani, 2002; Wang, 2003). In fact, RTP801/REDD1 is a direct target of HIF-1, a heterodimeric transcription factor that plays a pivotal role for survival in response to hypoxia (Shoshani, 2002). Furthermore, RTP801/REDD1 is controlled by p53 and p63 (Ellisen, 2002). Depending on the experimental setup and cell context, RTP801 overexpression either inhibits or increases apoptosis, suggesting a complex regulation and/or dependence on the developmental program of the cell (Reiling, 2004).
date revised: 20 July 2005
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